LOCUS       sequence-113664-        3426 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cloning of sgRNAs for expression in plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3426)
  AUTHORS   Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, 
            Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X,
            Dong Z, Liu YG
  TITLE     A robust CRISPR/Cas9 system for convenient, high-efficiency 
            multiplex genome editing in monocot and dicot plants.
  JOURNAL   Mol Plant. 2015 Apr 24. pii: S1674-2052(15)00204-X. doi: 
            10.1016/j.molp.2015.04.007.
  PUBMED    25917172
REFERENCE   2  (bases 1 to 3426)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Plant. 
            2015 Apr 24. pii: S1674-2052(15)00204-X. doi: 
            10.1016/j.molp.2015.04.007."
COMMENT     Sequence Label: pYLsgRNA-OsU3m/LacZ
FEATURES             Location/Qualifiers
     source          1..3426
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        20..95
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        364..746
                     /label=OsU3 promoter
                     /note="Oryza sativa (rice) snRNA U3 promoter"
     primer_bind     complement(780..796)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1005..1024)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1124..1146
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1184..1202)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1270..1374
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1375..1443,1444..2235)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1593..1612)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2406..2994
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2895..2914
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3148..3165
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3282..3303
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3318..3323,3324..3341,3342..3348)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3356..3372
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3361..3383
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3380..3396
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3380..3396
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ggatccagcg tgggtctcgg ttttagagct agaaatagca agttaaaata aggctagtcc
       61 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttcaagagct tggagtggat
      121 ggaattttcc tccgttttac ctgtggaatc ggcagcaaag gacgcgttga cattgtagga
      181 ctatattgct ctaataaagg aggcagctat gctggccgtc gttttacaac gtcgtgactg
      241 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
      301 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
      361 ctaaaggaat ctttaaacat acgaacagat cacttaaagt tcttctgaag caacttaaag
      421 ttatcaggca tgcatggatc ttggaggaat cagatgtgca gtcagggacc atagcacaag
      481 acaggcgtct tctactggtg ctaccagcaa atgctggaag ccgggaacac tgggtacgtt
      541 ggaaaccacg tgtgatgtga aggagtaaga taaactgtag gagaaaagca tttcgtagtg
      601 ggccatgaag cctttcagga catgtattgc agtatgggcc ggcccattac gcaattggac
      661 gacaacaaag gctagtatta gtaccacctc ggctatccac atagatcaaa gctggtttaa
      721 aagagttgtg cagatgatcc gtggcaagag acctctgaag ataacatact aagcttggca
      781 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
      841 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
      901 ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt
      961 acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat
     1021 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     1081 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     1141 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     1201 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     1261 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1321 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     1381 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     1441 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     1501 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     1561 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     1621 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1681 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1741 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1801 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1861 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1921 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1981 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     2041 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     2101 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     2161 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     2221 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     2281 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     2341 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2401 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2461 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2521 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     2581 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2641 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2701 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2761 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2821 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2881 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2941 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3001 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3061 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3121 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3181 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     3241 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     3301 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     3361 agcggataac aatttcacac aggaaacagc tatgaccatg attacgaatt cgagctcggt
     3421 acccgg
//
